All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
Phosphate (Pi) is an essential macronutrient for all living organisms. It is involved in most metabolic and signaling events in a cell, and is present in multiple cellular compartments. It serves various basic biological functions as a structural element in nucleic acids, phospholipids and ATP, as a metabolite involved in energy transfer, as a component in signal transduction cascades, and in the regulation of enzymes and metabolic processes.
Adenosine triphosphate (ATP) is the dominant ‘energy currency’ in the cell. The hydrolysis of ATP to adenosine diphosphate (ADP) plus Pi releases energy that fuels enumerable energy-requiring processes in the cell. Indeed, ATP is required for the phosphorylation of glucose (to generate glucose 6-phosphate), which enables glucose to enter the glycolytic pathway. Complete aerobic oxidation of a single glucose 6-phosphate molecule yields 30-36 molecules of ATP. Therefore, Pi is a critical metabolite and an essential nutrient, and the concentration of this molecule can profoundly alter cellular growth and metabolism. It is surprising, then, how little is known about the subcellular distribution phosphate and homeostasis under different phosphate concentrations.
To be able to measure phosphate levels directly in living cells, it would be useful to have a nanosensor for phosphate. A phosphate sensor would be an excellent tool for discovery and drug screening. The response of phosphate levels could be measured in real time in response to chemicals, metabolic events, transport steps, and signaling processes.
Recently a number of bacterial periplasmic binding proteins (PBP), which undergo a venus flytrap-like closure of two lobes upon substrate binding, have been successfully used as the scaffold of metabolite nanosensors (Fehr, M., Frommer, W. B., and Lalonde, S. (2002) Visualization of maltose uptake in living yeast cells by fluorescent nanosensors. Proc. Natl. Acad. Sci. USA 99, 9846-9851; Fehr, M., Lalonde, S., Lager, I., Wolff, M. W., and Frommer, W. B. (2003) In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors. J. Biol. Chem. 278, 19127-19133; Lager, I., Fehr, M., Frommer, W. B., and Lalonde, S. (2003) Development of a fluorescent nanosensor for ribose. FEBS Lett 553, 85-89). The PBP nanosensors thus far developed have been constructed using type I periplasmic binding proteins, wherein the fluorophores attached to the N- and C-termini of the protein are located on two different lobes.
There is a PBP for phosphate (PiBP) that has been isolated from various gram negative bacteria. For instance, the synthesis of the PiBP, the product of the pstS gene, is induced in E. coli when cell growth is limited by low Pi availability. However, in contrast to the type I PBPs used for nanosensors thus far, periplasmic phosphate binding protein has been classified as a type II PBP, with N- and C-termini located on the same protein lobe (Tam, R., and Saier, M. H. (1993) Microbiol Rev 57(2), 320-346; Fukami-Kobayashi, K., Tateno, Y., and Nishikawa, K. (1999) J Mol Biol 286(1), 279-290). The crystal structure of phosphate binding protein has been studied, and the modeled structures of PiBP also suggest a type II configuration although the assignment of both N- and C-terminal region is uncertain (Hirshberg, M., Henrick, K., Haire, L. L., Vasisht, N., Brune, M., Corrie, J. E. T., and Webb, M. R. (1998) Biochemistry-Us 37(29), 10381-10385; Ledvina, P. S., Tsai, A. L., Wang, Z. M., Koehl, E., and Quiocho, F. A. (1998) Protein Sci 7(12), 2550-2559). In addition, phosphate quenches fluorescence, making the analysis of phosphate sensors potentially problematic. Therefore, it was not clear whether a phosphate PBP sensor could be generated using the strategies employed for type I PBP sensors.